Inflammatory reaction is mainly due to the activities of inflammatory immune cells. In various inflammatory diseases, it is often observed that activated inflammatory immune cells influence neighboring fibroblast, epitherial cells and/or alpha smooth muscle cells to be activated into myoblast type cells. When fibroblast, epitherial cells, or alpha smooth muscle cells are activated into myoblast type cells, they show an enhanced ability for proliferation and migration and secret a large amount of extracellular matrix proteins such as fiber collagens which finally lead to induction of tissue fibrosis and hypertrophy. When this phenoma occur in wound healing process following tissue injury, the area of fibrosis gives scar in the tissue.
Recent researches provided with the evidence that cooperative activities of activated inflammatory cells with activated fibroblast, epitherial cells or alpha smooth muscle cells at the inflammatory site contribute importantly to various pathologies of inflammatory diseases.
One of the representative examples for the pathological phenomenon can be found in the wound healing process which occurs after external or internal injury on the tissues of our body by external phycal impact or internal continuous inflammation. After tissue injury, inflammatory immune cells such as macrophage, neutrophil, eosinophil, mast cell penetrate into the injured site rapidly and become activated to secret various cytokines, which in turn activate the neighboring fibroblast, epitherial cells, or alpha smooth muscle cells into myoblast type cells. These myoblasts synthesize and secret a large amount of extracellular matrix protein which leads to scar formation and tissue fibrosis and hypertrophy. (Gurtner G C et al., Trends Cell Biol. 15: 599-607, 2005). This pathological mechanism is the fundamental reason for scar formation in skin wound and for tissue fibrosis in lung, kidney and blood vessel. In addition, even in atopic dermatitis, asthma, COPD, psorisis, kelloid, proliferative retinopathy, the cooperative activation between inflammatory immune cells and fibrotic cells such as fibroblast, epitherial cells, or alpha smooth muscle cells occurs and these cells cooperatively contribute to the development of the pathology.
For example, atopic dermatitis is one of allergic skin inflammatory response where overactivation of type 2 helper T cell pathway is involved. In atopic dermatitis, IgE level in blood increases and cytokines such as IL-4, and IL-13 are also elevated. Water content in epidermis decreases and an excessive amount of mast cells are recruited into inflammatory site to secret a large amount of immune modulator by their degranulation. Interestingly collagen accumulation in skin is observed in atopic dermatitis. In psoriasis and kelloid, which are another skin inflammatory disease, the activation of keratinocyte is also involved in the process of tissue fibrosis along with invasion and activation of inflammatory cells.
Asthma is an allergic inflammatory reaction similarly to atopic dermatitis and happens in airway. Recently, it was known that activations of thoratic fibroblast, epitherial cells and/or alpha smooth muscle cells make an important contribution to inflammatory diseases in thoratic organs such as asthma and COPD in addition to inflammatory cells. In arterosclerosis, liver fibrosis, kidney fibrosis, and proliferative retinopathy, activations of alpha smooth muscle cells, liver stellate cell, kidney fibroblast, mesangial cell, ocular fibroblast respectively are observed along with the activations of inflammatory immune cells such as macrophage and they are importantly involved in the pathology of the inflammatory diseases.
When inflammatory immune cells are activated, an increased migration and secretion of various immune modulatory chemicals and cytokines are generally observed. For example, activation of macrophages increases their invasive activity through tissue induced by chemokines, the expression of iNOS (inducible nitric oxide synthase) and the synthesis of NO (nitric oxide). In addition, an increased synthesis of cytokines such as TNF-alpha is also typically observed. One of the notable features is the increased expression of alpha smooth muscle actin when Fibroblast, epitherial cells or alpha smooth muscle cells become activated into myoblast type cells. In addition, the cellular activity of FAK and Akt1 is increased, which contributes directly to an increase in cellular proliferation and migration.
We, inventors paid attention to the fact that the synergistic activation between inflammatory immune cells and neighboring fibroblast epitherial cell, or alpha smooth muscle cells occurs in various hardly-curable inflammatory diseases. Therefore we intended to develop compounds to suppress potently both the activation of immune cells and the activation of fibroblast, epitherial cells, or alpha smooth muscle cells into activated myoblast type cell in order to prevent and cure various hardly-curable inflammatory diseases or immune disorder. To accomplish this goal, the inventors intended to develop small molecular weight compounds to inhibit strongly against the activities of protein kinses involved directly in cell signaling pathways which lead the activations of inflammatory immune cells as well as fibroblast, epitherial cell, and alpha smooth muscle cells.
Especially c-Src family tyrosine kinases can be considered as the target for this goal among the cellular kinases since they play an important role in the signaling to activate various inflammatory immune cells. In addition, discoidin domain receptor family tyrosine kinases are important for the activations of fibroblast epitherial cells, or alpha smooth muscle cells to increase their proliferation and secretion of extra cellular matrix proteins.
Therefore, the inventors of the present invention provide with small molecule inhibitors to inhibit both c-Src family tyrosine kinases and discoidin domain receptor family tyrosine kinases simultaneously.
Protein kinases refer to proteins to catalyze the reaction to transfer a phosphate group at gamma position of ATP to hydroxyl group of tyrosine, serine or threonine at a specific substrate protein. The kinases to transfer phosphate to tyrosine to produce phospho-tyrosine are called protein tyrosine kinases. In vertebrate animal cells, more than 500 proteins kinases are found. One of their important roles is to play a role in signal transduction pathways which respond to the internal or external stimuli and their regulated activity is necessary for the maintenance of cell homiosis. However their excessive expression, over-activity or reduced expression or activity is directly or indirectly associated with various diseases such as cancer and immune disorders. For example, an abnormal over-activity of a certain protein kinases was proved as one of the main reasons for abnormal over-proliferations, migration, metastasis, and over-production of cytokines in cancer and immune cells.
Some protein tyrosine kinases are known as directly involved in the cell signaling to activate inflammatory immune cells and inflammatory response. Of them, Src family tyrosine kinase is one of the most extensively studied kinases.
Src family tyrosine kinases are consisted of total 8 non-receptor tyrosine kinases including Fgr, Fyn, Yes, Blk, Hck, Lck, Lyn along with c-Src. Their multiple in vivo functions are well elucidated and especially their important roles in immune cells to carry out immunological reactions are confirmed (Okutani et al., Am. J. Physiol. Lung Cell MoI. Physiol. 291: 129-141, 2006). Among the family proteins, Hck, Fgr, and Lyn are important for the activation of inflammatory cells such as macrophage and neutrophil and their adhesion in inflammatory site. Lck is known to be expressed mostly in T-cells and it is activated in the down stream of T-cell receptor in T cell signaling where its activity plays a critical role for the receptor signaling. In addition, Hck, Lyn, and Fgr showed an increased expression when monocyte cells and macrophage are activated by for example LPS (lipo-poly-saccharide). In addition, for example, when the expressions of Lyn, Fyn, Blk are inhibited, differentiation of immature B-cells into mature B-cells is suppressed in B-cell differentiation. Src family kinases are also indispensible for the accumulation of monocyte cells, macrophage, neutrophil cell in an inflammatory site and their activation as well as their participation to inflammatory reaction. Many cases of autoimmune and immunological diseases are associated with the activations of immune cells such as T-cell, B-cell, monocyte cell, and/or macrophage. In addition, other non-receptor tyrosine kinases are also important for the activations of inflammatory cells. Syk1 kinase is important for the activations of B cell, mastcell, macrophage, and monocyte cell. In addition, Btk1 is associated with the activations of macrophage, mast cell, and platelet. Therefore these three non-receptor tyrosine kinases of c-Src family, Syk1, Btk1 are considered as important major targets for developing drugs to suppress inflammatory reactions.
Discoidin domain receptor family (DDR) belongs to receptor tyrosine kinase family and consistes of type 1 and type 2 which have 89% homology in their kinase domain. These receptors have collagen as their activating ligand. DDR2 is important for cell migration, proliferation, and the increased synthesis of collagen when fibrblast cells are activated. It is also important for the activation of liver sellate cells which produce excessive collagen in liver cirrhosis, for the activation of synovial fibroblasts in joint tissue of arthritis and for the activation of arortic alpha smooth muscle cells of blood vessel wall in restenosis and arterosclerosis. DDR1 is also important for the proliferation of alpha smooth muscle cells in blood vessel and is involved in the tissue fibrosis by overactivation of fibroblast cells in kidney and lung. In addition, it plays a role for the accumulation of macrophage at inflammatory site in arterosclerosis, kidney fibrosis, and lung fibrosis.
We inventors synthesized small molecule compounds to have either thieno pyrimidine or thieno pyridine as a pharmacophore and have specific substituents around the pharmacophore to inhibit the activities of both c-Src family tyrosine kinases and discoidin domain receptor family tyrosine kinases simultaneously. Furthermore we confirmed that these compounds can inhibit both Src family tyrosine kinase activity that is important for the activation of cell signaling in immune cells and discoidin domain receptor family tyrosine kinase activity that is involved in the activations of fibroblast and alpha smooth muscle cells. Therefore we accomplished the present invention by confirming that these compounds are useful to cure various inflammatory diseases or immune disorder and to promote wound healing, and suppression of scar formation.